Synthesis and Evaluation of 177Lu-DOTA-PD-L1-i and 225Ac-HEHA-PD-L1-i as Potential Radiopharmaceuticals for Tumor Microenvironment-Targeted Radiotherapy

Current cancer therapies focus on reducing immunosuppression and remodeling the tumor microenvironment to inhibit metastasis, cancer progression, and therapeutic resistance. Programmed death receptor 1 (PD-1) is expressed on immune T cells and is one of the so-called checkpoint proteins that can suppress or stop the immune response. To evade the immune system, cancer cells overexpress a PD-1 inhibitor protein (PD-L1), which binds to the surface of T cells to activate signaling pathways that induce immune suppression. This research aimed to synthesize PD-L1 inhibitory peptides (PD-L1-i) labeled with lutetium-177 (177Lu-DOTA-PD-L1-i) and actinium-225 (225Ac-HEHA-PD-L1-i) and to preclinically evaluate their potential as radiopharmaceuticals for targeted radiotherapy at the tumor microenvironment level. Using PD-L1-i peptide as starting material, conjugation with HEHA-benzene-SCN and DOTA-benzene-SCN was performed to yield DOTA-PD-L1-i and HEHA-PD-L1-I, which were characterized by FT-IR, UV-vis spectroscopy, and HPLC. After labeling the conjugates with 225Ac and 177Lu, cellular uptake in HCC827 cancer cells (PD-L1 positive), conjugate specificity evaluation by immunofluorescence, radiotracer effect on cell viability, biodistribution, biokinetics, and assessment of radiation absorbed dose in mice with in duced lung micrometastases were performed. 225Ac-HEHA-PD-L1-i and 177Lu-DOTA-PD-L1-i, obtained with radiochemical purities of 95 ± 3% and 98.5 ± 0.5%, respectively, showed in vitro and in vivo specific recognition for the PD-L1 protein in lung cancer cells and high uptake in HCC287 lung micrometastases (>30% ID). The biokinetic profiles of 177Lu-DOTA-PD-L1-i and 225Ac-DOTA-PD-L1-i showed rapid blood clearance with renal and hepatobiliary elimination and no accumulation in normal tissues. 225Ac-DOTA-PD-L1-i produced a radiation dose of 5.15 mGy/MBq to lung micrometastases. In the case of 177Lu-DOTA-PD-L1-i, the radiation dose delivered to the lung micrometastases was ten times (43 mGy/MBq) that delivered to the kidneys (4.20 mGy/MBq) and fifty times that delivered to the liver (0.85 mGy/MBq). Therefore, the radiotherapeutic PD-L1-i ligands of 225Ac and 177Lu developed in this research could be combined with immunotherapy to enhance the therapeutic effect in various types of cancer.


Introduction
Tumors are pathological tissues that comprise cancer cells and the tumor microenvironment (TME). The TME includes endothelial and immune cells communicating in a dynamic network through messenger molecules and acellular factors, mainly chemokines, inflammatory enzymes, growth factors, cytokines, and extracellular matrix components [1]. The function of immune cells and messenger molecules associated with the TME is a relevant topic in the research field of cancer biology. 2 of 19 Therefore, current cancer therapies converge on inhibiting cancer progression and therapeutic resistance by reducing immunosuppression and remodeling the TME [2,3]. In this context, previous research on the development of imaging radioligands targeting proteins of the tumor microenvironment, such as nanobodies against proteins of the extracellular matrix [4], radioligands to target the chemokine receptor-4 (CXCR-4) [5][6][7], fibroblast activation protein (FAP) radioinhibitors [8][9][10][11][12], and radiotracers directed to integrins and the prostate-specific membrane antigen (PSMA) expressed in the tumor neovasculature [13][14][15][16][17][18][19], among others, have proven to be helpful in detecting spatial and temporal changes in the tumor microenvironment phenotype of different cancer entities, which have been essential in defining the treatment of patients according to the expression or suppression of proteins involved in the disease. In particular, the immunotherapeutic pathway has been exploited with various agents, such as monoclonal antibodies, radioimmunoconjugates, and nano-immune systems [20,21].
Immune checkpoint inhibitor (ICI) therapy is an important treatment option for various cancers [22,23]. Among the targeted immune checkpoints, programmed death receptor 1 (PD-1) and its ligand (PD-L1) are the key molecular targets for ICI therapy. Expressed on immune T cells, PD-1 is one of the checkpoint molecules that can suppress or stop immune responses [22]. Therefore, to evade the immune system, cancer cells overexpress a PD-1 inhibitor protein (PD-L1) that binds to the surface of T cells to activate signaling pathways that induce immune suppression. Thus, PD-L1 is a reproducible biomarker that allows therapeutic decisions to be made and the success of the treatment to be monitored. PD-L1 gene expression imaging, which is highly dynamic, can be monitored in a highly specific manner by SPECT/CT and PET/CT imaging techniques, which are used to determine whether a patient is a candidate for immunotherapeutic treatment with anti-PD-L1 or anti-PD-1 antibodies [24][25][26][27]. However, PD-L1 inhibitor radiotherapeutic peptides have not yet been investigated. Lutetium-177 is a β-emitting radionuclide with a mean tissue penetration of 0.67 mm (maximum energy: 497 keV), and actinium-225 is an alpha-emitting radionuclide with a maximum tissue penetration of a few cell diameters (emission of four high-energy alpha particles with an energy of 6 MeV). Therefore, both radionuclides have sufficient energy and tissue penetration to kill target tumor cells with limited effect on adjacent healthy cells.
This research aimed to synthesize lutetium-177-and actinium-225-labeled PD-L1 inhibitory peptides ( 177 Lu-DOTA-PD-L1-i and 225 Ac-HEHA-PD-L1-i) and to preclinically evaluate their potential as radiopharmaceuticals for targeted radiotherapy of various cancers at the tumor microenvironment level.  (Figure 1). The PD-L1 inhibitor ligands (PD-L1-i, DOTA-PD-L1-i, and HEHA-PD-L1-i) were also characterized using FT-IR, UV-vis spectroscopy, and HPLC, as described below. The PD-L1 inhibitor ligands (PD-L1-i, DOTA-PD-L1-i, and HEHA-PD-L1-i) were als characterized using FT-IR, UV-vis spectroscopy, and HPLC, as described below. The IR analysis of the PD-L1 inhibitor conjugates showed that the band at 1658 cm of PD-L1-i, associated with the -C=O stretching of carbonyl bonds (-CO-NH-), decrease in energy to 1671 cm −1 in both DOTA-PD-L1-i and HEHA-PD-L1-i as a result of the conju gation reaction (-HN-CS-NH-benzene-DOTA/-HEHA), which produced a decrease i the absorption frequency, as dipole moments in the cyclic peptide ring were reduced (Fig  ure 2). On the other hand, the band at 3310 cm −1 of PD-L1-I, associated with the primar amine of ornithine (-NH2), increased in energy to 3276 cm −1 and 3280 cm -1 in the DOTA PD-L1-i and HEHA-PD-L1-i conjugates, respectively, which is characteristic of -NH bond formation (-HN-CS-). Further evidence for the conjugation process was the sub stantial increase in the band's intensity at 1202 cm −1 associated with a rise in the -C-O stretching from DOTA and HEHA carboxylic acids (O=CO-H) ( Figure 2). The IR analysis of the PD-L1 inhibitor conjugates showed that the band at 1658 cm −1 of PD-L1-i, associated with the -C=O stretching of carbonyl bonds (-CO-NH-), decreased in energy to 1671 cm −1 in both DOTA-PD-L1-i and HEHA-PD-L1-i as a result of the conjugation reaction (-HN-CS-NH-benzene-DOTA/-HEHA), which produced a decrease in the absorption frequency, as dipole moments in the cyclic peptide ring were reduced ( Figure 2). On the other hand, the band at 3310 cm −1 of PD-L1-I, associated with the primary amine of ornithine (-NH 2 ), increased in energy to 3276 cm −1 and 3280 cm −1 in the DOTA-PD-L1-i and HEHA-PD-L1-i conjugates, respectively, which is characteristic of -NH-bond formation (-HN-CS-). Further evidence for the conjugation process was the substantial increase in the band's intensity at 1202 cm −1 associated with a rise in the -C-Ostretching from DOTA and HEHA carboxylic acids (O=CO-H) ( Figure 2).  The UV-vis spectra of the PD-L1-i conjugates show several shoulders and peak 211 nm to 226 nm, which are assigned to the n→σ* electronic transitions from sulf heteroatom (-H2C-S-CH2 -), π→π* electronic transitions from double bonds within acid carbonyls, and -C=O from carboxylic acids. The band at 283 nm is associate π→π* and n→π* electronic transitions (-C=C-and -C=N-groups) of the peptid chain rings (e.g., imidazole ring) ( Figure 3B). The single broad and well-defined b 283 nm of DOTA-PD-L1-i and HEHA-PD-L1-i appears different from that of the p sors (278 nm from the PD-L1-i ligand and the 272 nm and 281 nm doublet from the D /HEHA-Benzene-SCN precursors), indicating new chemical compound formatio L1-i conjugates) (Figure 3). The UV-vis spectra of the PD-L1-i conjugates show several shoulders and peaks from 211 nm to 226 nm, which are assigned to the n→σ* electronic transitions from sulfur as a heteroatom (-H 2 C-S-CH 2 − ), π→π* electronic transitions from double bonds within amino acid carbonyls, and -C=O from carboxylic acids. The band at 283 nm is associated with π→π* and n→π* electronic transitions (-C=C-and -C=N-groups) of the peptide side chain rings (e.g., imidazole ring) ( Figure 3B). The single broad and well-defined band at 283 nm of DOTA-PD-L1-i and HEHA-PD-L1-i appears different from that of the precursors (278 nm from the PD-L1-i ligand and the 272 nm and 281 nm doublet from the DOTA-/HEHA-Benzene-SCN precursors), indicating new chemical compound formation (PD-L1-i conjugates) (Figure 3).

Radiolabeling of PD-L1-i Ligands with 177 Lu and 225 Ac
The radiopharmaceuticals 177 Lu-DOTA-PD-L1-i and 225 Ac-HEHA-PD-L1-i were initially obtained with radiochemical purities of 98.5 ± 0.5% (without further purification) and 83 ± 2% (95 ± 3% after purification using Sep-Pak C18 cartridges), respectively, as determined by radio-HPLC (C-18 reversed phase) (Figures 4 and 5). However, to assess the radiochemical purity of 225 Ac-HEHA-PD-L1-i, fractions of 1 mL eluted from the column had to be collected and counted in a NaI(Tl) detector ( Figure 5). Retention times for 177 LuCl3 and 225 AcCl3 were between 2.5 and 4.5 min and 16 to 18 min for 177 Lu-DOTA-PD-L1-i and 225 Ac-HEHA-PD-L1-i. It is important to note that chromatograms are usually achieved with radiometric and UV-vis detectors. After injecting the radiotracer into the HPLC system, it was first identified using the UV-vis instrument, and after 0.8-1.0 min (0.8-1.0 mL, 1 mL/min), it was detected with the radiometric detector. An agreement of retention times between the peak observed in the UV-vis chromatogram (PD-L1-i conjugate) and the radio-chromatogram is usually considered evidence of the chemical identity of the radiopharmaceutical (Figure 4) [29].

Radiolabeling of PD-L1-i Ligands with 177 Lu and 225 Ac
The radiopharmaceuticals 177 Lu-DOTA-PD-L1-i and 225 Ac-HEHA-PD-L1-i were initially obtained with radiochemical purities of 98.5 ± 0.5% (without further purification) and 83 ± 2% (95 ± 3% after purification using Sep-Pak C18 cartridges), respectively, as determined by radio-HPLC (C-18 reversed phase) (Figures 4 and 5). However, to assess the radiochemical purity of 225 Ac-HEHA-PD-L1-i, fractions of 1 mL eluted from the column had to be collected and counted in a NaI(Tl) detector ( Figure 5). Retention times for 177 LuCl 3 and 225 AcCl 3 were between 2.5 and 4.5 min and 16 to 18 min for 177 Lu-DOTA-PD-L1-i and 225 Ac-HEHA-PD-L1-i. It is important to note that chromatograms are usually achieved with radiometric and UV-vis detectors. After injecting the radiotracer into the HPLC system, it was first identified using the UV-vis instrument, and after 0.8-1.0 min (0.8-1.0 mL, 1 mL/min), it was detected with the radiometric detector. An agreement of retention times between the peak observed in the UV-vis chromatogram (PD-L1-i conjugate) and the radio-chromatogram is usually considered evidence of the chemical identity of the radiopharmaceutical ( Figure 4) [29].

Stability
PD-L1 inhibitor radioconjugates showed no differences regarding stability as a function of the dilution medium. 177 Lu-DOTA-PD-L1-i remained stable in water (injectablegrade water/ascorbic acid/sodium acetate) and human serum with radiochemical purities greater than 97.5 ± 0.5% after 72 h of labeling (inset: Figure 4). In contrast, the radiochemical purity of 225 Ac-HEHA-PD-L1-i decreased to 64 ± 4% in both water and human serum 72 h after purification, at a rate of approximately 0.388%/h (slope = −0.388) (inset: Figure 5). It is important to mention that, although it was not the aim of this study, we also examined the preparation of 225 Ac-DOTA-PD-L1-i, which showed a radiochemical purity of 9 ± 3% in water after 72 h of labeling (1.149%/h rate of decrease). It is well documented that the main disadvantage of 225 Ac complexes is the recoil energy of the nucleus during decay, which induces the breaking of the bond with the chelator [30]. In agreement with previous research, the use of HEHA instead of DOTA improves the stability of the coordination complex [31,32]. Nevertheless, the recoil energy of 225 Ac and its progeny (four alpha particles) will inevitably produce, to a greater or lesser extent, time-unstable 225 Ac complexes [30,33].

Cellular Uptake Assay
HCC827 cells (PD-L1 positive control) were found to uptake approximately fourteen times more 177 Lu-DOTA-PD-L1-i and 225 Ac-HEHA-PD-L1-i than C6 cells (PD-L1 negative control) (Figure 6a). The expression of PD-L1 in HCC827 cells ( Figure 6b) and the lack of PD-L1 expression in C6 cells ( Figure 6c) were confirmed by immunofluorescent microscopy, thus ensuring that the radiotracer uptake is specific and associated with the presence of PD-L1 in HCC827 cells. It was also proven by immunofluorescent microscopy that the PD-L1-i ligand, used in this research for 177 Lu and 225 Ac labeling, is recognized by the PD-L1 protein expressed in HCC827 cells ( Figure 7). times more 177 Lu-DOTA-PD-L1-i and 225 Ac-HEHA-PD-L1-i than C6 cells (PD-L1 negative control) (Figure 6a). The expression of PD-L1 in HCC827 cells ( Figure 6b) and the lack of PD-L1 expression in C6 cells ( Figure 6c) were confirmed by immunofluorescent microscopy, thus ensuring that the radiotracer uptake is specific and associated with the presence of PD-L1 in HCC827 cells. It was also proven by immunofluorescent microscopy that the PD-L1-i ligand, used in this research for 177 Lu and 225 Ac labeling, is recognized by the PD-L1 protein expressed in HCC827 cells ( Figure 7).

Cell Viability Assays
A decrease in the viability of HCC827 cells was observed depending on the targetspecific radiotherapy mediated by the PD-L1 recognition ( Figure 8). Cells treated with an acute radiation dose of 15 Gy using 177 Lu-DOTA-PD-L1-i (cells incubated for 5 h with 177 Lu-DOTA-PD-L1-i; 1.4 MBq/20,000) showed a net decrease viability of 31.42 ± 2.01%, with a statistically significant difference (p < 0.05, Student's t-test) compared to the group treated with PD-L1-i (decrease viability of 4 ± 1%; 0.1 µg/20,000 cells) (Figure 8b,c). The viability of cells treated with 5 Gy of 225 Ac-HEHA-PD-L1-i (cells incubated for 5 h with 225 Ac-HEHA-PD-L1-i; 0.37 kBq/20,000 cells) had a net decrease viability of 42.54 ± 3.87% vs. 4 ± 1% of those treated with PD-L1-i (Figure 8b,d). The reason why we used a three times lower radiation dose for 225 Ac-HEHA-PD-L1-i (5 Gy) than for 177 Lu-DOTA-PD-L1-i (15 Gy) was the consideration of the high number of short path ionizations generated by alpha particle emitters (high LET) damaging DNA structures. This effect, known as RBE (relative biological efficiency), explains why actinium-225 affected cell viability more than lutetium-177, even at a lower radiation dose [34].

Cell Viability Assays
A decrease in the viability of HCC827 cells was observed depending on the targetspecific radiotherapy mediated by the PD-L1 recognition ( Figure 8). Cells treated with an acute radiation dose of 15 Gy using 177 Lu-DOTA-PD-L1-i (cells incubated for 5 h with 177 Lu-DOTA-PD-L1-i; 1.4 MBq/20,000) showed a net decrease viability of 31.42 ± 2.01%, with a statistically significant difference (p < 0.05, Student's t-test) compared to the group treated with PD-L1-i (decrease viability of 4 ± 1%; 0.1 µg/20,000 cells) (Figure 8b,c). The viability of cells treated with 5 Gy of 225 Ac-HEHA-PD-L1-i (cells incubated for 5 h with 225 Ac-HEHA-PD-L1-i; 0.37 kBq/20,000 cells) had a net decrease viability of 42.54 ± 3.87% vs. 4 ± 1% of those treated with PD-L1-i (Figure 8b,d). The reason why we used a three times lower radiation dose for 225 Ac-HEHA-PD-L1-i (5 Gy) than for 177 Lu-DOTA-PD-L1-i (15 Gy) was the consideration of the high number of short path ionizations generated by alpha particle emitters (high LET) damaging DNA structures. This effect, known as RBE (relative biological efficiency), explains why actinium-225 affected cell viability more than lutetium-177, even at a lower radiation dose [34].

Biodistribution and Biokinetic Models
In healthy mice, the 177 Lu-DOTA-PD-L1-i and 225 Ac-DOTA-PD-L1-i biodistribution profiles showed rapid blood clearance with renal and hepatobiliary elimination and no accumulation in normal tissues. (Figures 9a and 10a).

In Vivo Evaluation 2.4.1. Biodistribution and Biokinetic Models
In healthy mice, the 177 Lu-DOTA-PD-L1-i and 225 Ac-DOTA-PD-L1-i biodistribution profiles showed rapid blood clearance with renal and hepatobiliary elimination and no accumulation in normal tissues. (Figures 9a and 10a).  (Figure 9b); consequently, the absorbed radiation dose delivered to the lungs was ten times that given to the kidneys and fifty times than that to the liver (Table 1).  According to the biokinetic model of 177 Lu-DOTA-PD-L1-i obtained in mice intravenously injected with HCC827 lung cancer cells (PD-L1+) (Figure 9b), seventy-four percent of the activity taken up in the liver was cleared rapidly (λ = 1.23), with a half-life of 0.56 h (t1/2 = ln2/λ = 0.56 h) associated with the hepatobiliary elimination, while twenty-six percent was cleared slowly (λ = 0.01; t1/2 = 69 h), possibly related to the fraction retained in the reticuloendothelial system. The short half-life time observed in the biokinetic model of kidneys (t1/2 = ln2/3.65 = 0.19 h) can be correlated with renal elimination and reabsorption in the proximal tubule, whereas the slow elimination slope (t1/2 = ln2/0.009 = 77 h) would be related to the breakdown of the peptide fragment, where 177 Lu is retained for a longer time [35]. The most extended 177 Lu-DOTA-PD-L1-i retention time was observed in lungs invaded with HCC827 cancer cells. Ninety percent of the lung uptake activity had a halflife of 346.5 h, and only ten percent had a half-life of 17.3 h ( Figure 9b); consequently, the absorbed radiation dose delivered to the lungs was ten times that given to the kidneys and fifty times than that to the liver (Table 1). According to the biokinetic model of 177 Lu-DOTA-PD-L1-i obtained in mice intravenously injected with HCC827 lung cancer cells (PD-L1+) (Figure 9b), seventy-four percent of the activity taken up in the liver was cleared rapidly (λ = 1.23), with a half-life of 0.56 h (t 1/2 = ln2/λ = 0.56 h) associated with the hepatobiliary elimination, while twenty-six percent was cleared slowly (λ = 0.01; t 1/2 = 69 h), possibly related to the fraction retained in the reticuloendothelial system. The short half-life time observed in the biokinetic model of kidneys (t 1/2 = ln2/3.65 = 0.19 h) can be correlated with renal elimination and reabsorption in the proximal tubule, whereas the slow elimination slope (t 1/2 = ln2/0.009 = 77 h) would be related to the breakdown of the peptide fragment, where 177 Lu is retained for a longer time [35]. The most extended 177 Lu-DOTA-PD-L1-i retention time was observed in lungs invaded with HCC827 cancer cells. Ninety percent of the lung uptake activity had a halflife of 346.5 h, and only ten percent had a half-life of 17.3 h (Figure 9b); consequently, the absorbed radiation dose delivered to the lungs was ten times that given to the kidneys and fifty times than that to the liver (Table 1).  In the case of the 225 Ac-HEHA-PD-L1-i radiocomplex, the biodistribution profile during the first hours after injection showed higher uptake of radioactivity in most of the organs examined and a significantly higher hepatic uptake and retention (17.4% of the injected dose was eliminated with a half-life of 69.3 h) than in the case of the 177 Lu-DOTA-PD-L1-i complex, which may be related to the presence of Ac 3+ and its progeny released from the radiocomplex, initially distributed in healthy organs and subsequently retained in the liver and, to a lesser extent, in the kidney (12.6% of the injected dose was eliminated with a half-life of 0.19 h and 8.14% with a half-life of 0.27 h) ( Figure 10). Therefore, the absorbed radiation dose delivered to the lungs was almost equal to that of the kidneys and only 3.5 times that given to the liver (Table 1). Although biodistribution was performed for dosimetric purposes (only %ID in whole organs was calculated), the Ac-225 activity present in bone was also measured and was not significant (0.3 ± 0.2% ID/g).

Radioisotopic Imaging
In vivo and ex vivo radioisotopic images of athymic mice with lung micrometastases after 177 Lu-DOTA-PD-L1-i administration showed that the only tissue that accumulated the radiotracer was the lung invaded by HCC827 cancer cells, while the liver and kidneys captured and eliminated the radiotracer according to their physiological excretory function ( Figure 11). In the case of the 225 Ac-HEHA-PD-L1-i radiocomplex, the biodistribution profile during the first hours after injection showed higher uptake of radioactivity in most of the organs examined and a significantly higher hepatic uptake and retention (17.4% of the injected dose was eliminated with a half-life of 69.3 h) than in the case of the 177 Lu-DOTA-PD-L1-i complex, which may be related to the presence of Ac 3+ and its progeny released from the radiocomplex, initially distributed in healthy organs and subsequently retained in the liver and, to a lesser extent, in the kidney (12.6% of the injected dose was eliminated with a half-life of 0.19 h and 8.14% with a half-life of 0.27 h) ( Figure 10). Therefore, the absorbed radiation dose delivered to the lungs was almost equal to that of the kidneys and only 3.5 times that given to the liver (Table 1). Although biodistribution was performed for dosimetric purposes (only %ID in whole organs was calculated), the Ac-225 activity present in bone was also measured and was not significant (0.3 ± 0.2% ID/g).

Radioisotopic Imaging
In vivo and ex vivo radioisotopic images of athymic mice with lung micrometastases after 177 Lu-DOTA-PD-L1-i administration showed that the only tissue that accumulated the radiotracer was the lung invaded by HCC827 cancer cells, while the liver and kidneys captured and eliminated the radiotracer according to their physiological excretory function ( Figure 11).  In addition, paraffin sections of the lungs of mice injected with HCC827 cells were analyzed for growth within the lung parenchyma using hematoxylin and eosin histological staining (Figure 12e). Metastases were found in the lungs of all mice injected with cancer cells, as tumors grew within the lung parenchyma in the form of atypical nuclei and mitotic structures (Figure 12e). No metastases were found in the lungs of untreated control animals (control: without induced lung micrometastases) (Figure 12f). The metastatic adenocarcinoma tissue growing within the lung parenchyma distinctly differed from normal alveolar lung tissue and demonstrated biphasic growth of this cancerous tissue. Histological staining (H&E) analysis also revealed no pathologic changes in the liver and kidney of mice injected with the radiopharmaceuticals ( Figure A4). There was no presence of any form of steatosis, cytoplasmic changes, or necrotic evidence. Hemorrhage and inflammatory infiltrations were absent ( Figure A4). No evidence of cytotoxic degeneration was observed in nonparenchymal cells or hepatocytes ( Figure A4). No significant cytotoxic effect was observed in the function of liver and kidney involved in metabolism, elimination, and or excretion of 177 Lu-PD-L1-i and 225 Ac-HEHA-PD-L1-i (Table A1). The biodistribution of the radiopharmaceuticals analyzed in this study suggests tha 177 Lu-PD-L1-i may have a better biokinetic and dosimetric profile than 225 Ac-PD-L1-i fo use in targeted radiotherapy. Therefore, the results obtained justify that, as future work preclinical studies of therapeutic efficacy should be carried out not only in a lung cance The 177 Lu-DOTA-PD-L1-i uptake pattern (average number of emitted photons per normalized area showed in the color bar of Figure 12) in the lungs of mice with metastatic HCC827 cells (298 ± 82 photons/cm 2 ) was statistically significantly different (p < 0.05; Student's t-test) compared with the group of mice in which metastases were not induced (78 ± 32 photons/cm 2 ).
Immune checkpoint inhibitor (ICI) therapy using nanobodies, minibodies, antibodies, and, more recently, PD-L1 or PD-1 inhibitory peptides for nuclear molecular imaging have demonstrated their utility as highly specific biomolecules for targeting the tumor microenvironment (TME) [27]. Therefore, the radiotherapeutic PD-L1-i ligands of 225 Ac and 177 Lu developed in this research, with biokinetic and dosimetric properties suitable for delivering ablative radiation doses at the TME level, can potentially be combined with ICI immunotherapy and enhance the therapeutic effect in different types of cancer. It has been previously reported, at the preclinical and clinical levels, that radioimmunotherapy improves therapeutic outcomes, mitigates immune side effects, and reduces the induced radio-toxicity [36] in addition to promoting tumor regression of nonirradiated metastases in patients (abscopal effect), due to the power of radiation on increased cancer cell apoptosis and release of tumor-associated antigens with subsequent delivery of CD8+ T cells as an anti-tumor response [37]. However, poorly immunogenic tumors do not respond to ICI therapy. In these cases, the use of 225 Ac-HEHA-PD-L1-i and 177 Lu-DOTA-PD-L1-i in combination with the nonradiolabeled PD-L1-i ligand would be a viable option. This proposal is based on recent successful therapeutic results obtained by combining the radioligand 177 Lu-PSMA-617 or 225 Ac-PSMA-617 (targeting prostate cancer cells) with the pharmaceutical anti-PD-1 (pembrolizumab), which showed a marked synergistic effect and overcame immune limitations in patients with poorly immunogenic tumors [38].
The biodistribution of the radiopharmaceuticals analyzed in this study suggests that 177 Lu-PD-L1-i may have a better biokinetic and dosimetric profile than 225 Ac-PD-L1-i for use in targeted radiotherapy. Therefore, the results obtained justify that, as future work, preclinical studies of therapeutic efficacy should be carried out not only in a lung cancer model because, given the nature of PD-L1 gene expression in a wide range of cancer types, therapeutic efficacy should be evaluated in different models and cancer types induced and correlated with other markers of immune response.
The UV-vis spectra (Thermo Fisher Genesys 10S spectrometer) of the PD-L1-i conjugates (1 mg/mL) were obtained from 210 to 300 nm. Finally, the mixture was incubated at 95 • C for 60 min. After labeling, 225 Ac-HEHA-PD-L1-I was purified using solid-phase extraction (Speak C18 cartridge, Waters). Radioactive solutions were diluted in injectable-grade water (1 mL) for further use. For comparative purposes, Lu-DOTA-PD-L1-i was also prepared under the same conditions using stable LuCl 3 (anhydrous powder; 99.99% trace metal basis; Sigma-Aldrich: Saint Louis, MO, USA) followed by HPLC purification.

Cellular Uptake
HCC827 human lung cancer (PD-L1 positive) and C6 mouse glioma (PD-L1 negative) cell lines were cultured in RPMI-1640 medium with penicillin and streptomycin (100 µg/mL) and fetal bovine serum (15%) in 5% CO 2 at 37 • C. Cells diluted in PBS (pH 7) (1 × 10 5 cells/tube) received two different treatments: (a) 177 Lu-DOTA-PD-L1-i (4 kBq) (n = 3) and (b) 225 Ac-HEHA-PD-L1-i (0.04 kBq) (n = 3). Cells were incubated with each treatment at 37 • C for 1 h. After incubation, tubes were measured in a NaI(Tl) detector to determine the initial activity (100%). The tubes were centrifuged at 500× g for 10 min. Then, a mixture of acetic acid/0.5 M NaCl was added, and the centrifugation was repeated. The liquid was removed, and the button activity was measured, corresponding to the percentage of activity captured by the cells regarding the initial activity.

Cell Viability Assay
The effect of 177 Lu DOTA-PD-L1-i, 225 Ac-HEHA-PD-L1-i, and PD-L1-i on HCC827 cell viability (5 × 10 4 ) was determined by flow cytometry with the Muse ® Count & Viability Kit from Luminex, DiaSorin, USA. Before taking readings with the Muse Cell Analyzer (Merck Millipore, Burlington, MA, USA) previously calibrated with the Muse ® System Check kit, the baseline detection was established using a fresh sample of untreated cells that served to delimit the size and granularity of the cell line, as well as the basal viability of the population. Also, the reading window for dead cells was established using an aliquot of the same population treated with 0.5% Triton-X100. Once the detection window for the HCC827 cells was established to discriminate between live and dead cells, 50 µL (2 × 10 4 cells/200 µL) were taken from the cell suspension that had been treated with cold ligand (PD-L1-i, 0.1 µg), with 1.4 MBq of 177 Lu DOTA-PD-L1-i or 0.37 kBq of 225 Ac-HEHA-PD-L1-i for 5 h and then mixed with 450 µL of the viability reagent; after 5 min incubation at room temperature and in the dark, the samples were read in triplicate. For the calculation of the radiation absorbed dose to cell nuclei, the total number of nuclear transformations (N) normalized to the unit of administered activity (Bq . s/Bq) was obtained with the A(t) mathematical integration (from t = 0 to t = 5 h). Multiplying N by the dose factor (DF)(Gy/Bq.s) from the cytoplasm to the nucleus (MIRDcell software, version 2.1), the radiation absorbed dose was obtained. The progeny of 225 Ac was considered. The model included cells with a diameter of 20 µm, a nucleus radius of 3 µm, and a density of 1 mg/mL.

Biodistribution and Studies
Under an approved protocol (No. 07-2018-2021), all animal procedures followed the ethical regulations for handling laboratory animals (NOM-062-ZOO-1999) and the Institutional Animal Care and Use Committee requirements. Male Nu/Nu (UPEAL, CINVESTAV, I.PN., Mexico City, Mexico) mice aged 6-8 weeks were maintained in a pathogen-free barrier facility. Mice were injected with 3.7 MBq (50 µL) of 177 Lu DOTA-PD-L1-I or 3.7 kBq (50 µL) of 225 Ac-HEHA-PD-L1-i in the tail vein and sacrificed at 0.5, 1, 3, 24, 72, and 96 h (n = 3). The heart, lungs, liver, pancreas, spleen, kidneys, small intestine, brain, and stomach were dissected to be measured in a NaI(Tl) radioactivity detector. The results are expressed as the percentage of injected dose (%ID) per organ. Blood samples were also extracted, and the activity was recorded as %ID/g. A second group of mice was inoculated intravenously with 1 × 10 6 HCC827 cells. The animals were treated with 3.7 MBq (50 µL) of 177 Lu DOTA-PD-L1-i (n = 3) or 3.7 kBq (50 µL) of 225 Ac-HEHA-PD-L1-i (n = 3) via tail vein injection on day five post-tumor cell inoculation. The animals were dissected as described above. In the case of 177 Lu DOTA-PD-L1-i, radioisotopic/X-ray in vivo/ex vivo images were obtained in a Preclinical Imaging System (Bruker, XTREME). For in vivo imaging, mice were anesthetized with oxygen and 2% isoflurane.

Absorbed Radiation Dose Assessment
The %ID values were used to obtain the biokinetic models (q h (t)). The A h (t) functions were obtained by decay-correcting the biokinetic models, that is, by adding to the biological constant (λ B ) the radioactive constant (λ R ), as follows (Equation (1)): For dosimetry, 225 Ac decay chain was considered. The biokinetic models' integration (from t = 0 to t = ∞) calculated the N value in each murine organ. The DF values were those reported in the OLINDA 2.0 code.

Histopathological Assessment
Healthy lungs, kidneys, liver, and lungs from mice injected i.v. with HCC827 cells were fixed with 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin, and sectioned in 4-5 µm thick sections using a microtome. Paraffin sections of lung samples were deparaffinized in xylene and rehydrated in a series of graded alcohols. Dewaxed tissue sections were stained with hematoxylin and eosin (H&E). The sections were assessed using a light microscope (magnification of 100x) (Zeiss; Mod. Axioscope: Oberkochen, Germany). Images were acquired with a digital camera (5 MP high-speed color; AmScope; Mod.Mu500: Irvine, CA, USA).

Evaluation of Creatinine and Liver Enzyme Levels
Mouse blood samples collected at 96 h after radiopharmaceutical administration were used for the determination of alanine aminotransferase (ALT), creatinine, and aspartate aminotransferase (AST). Creatinine was measured titrimetrically. The picrate method was used. AST and ALT were quantified using a Roche Diagnostics kit for UV assays.

Conclusions
The radiotherapeutic PD-L1-i ligands of 225 Ac and 177 Lu developed in this research, with biokinetic and dosimetric properties suitable for delivering ablative radiation doses at the tumor microenvironment level, could potentially be combined with immune checkpoint inhibitor therapy and enhance the therapeutic effect in various types of cancer. Considering the ease of preparation and high in vitro and in vivo stability, 177 Lu-DOTA-PD-L1-i is proposed as the best option for in vivo PD-L1 protein-targeted radiotherapy.

Appendix A
Acknowledgments: This study was conducted as a component of the activities of the "Laboratorio Nacional de Investigación y Desarrollo de Radiofármacos, ININ". The authors thank the "Laboratorio Nacional de Investigaciones en Forense Nuclear", CONACyT (LANAFONU), for conducting the spectroscopy analyses.   Acknowledgments: This study was conducted as a component of the activities of the "Laboratorio Nacional de Investigación y Desarrollo de Radiofármacos, ININ". The authors thank the "Laboratorio Nacional de Investigaciones en Forense Nuclear", CONACyT (LANAFONU), for conducting the spectroscopy analyses.

Conflicts of Interest:
The authors declare no conflict of interest.
Appendix A Figure A1. Schematic chemical structure of the PD-L1-i ligand.    Figure A4. Microscopic views (×40) of kidney and liver tissues from mice exposed to 177 Lu-DOTA-PD-L1-i and 225 Ac-HEHA-PD-L1-i. Histological staining (H&E) analysis revealed no pathologic changes in the liver and kidney. There was no presence of any form of steatosis, cytoplasmic changes, or necrotic evidence. Hemorrhage and inflammatory infiltrations were absent. No evidence of cytotoxic degeneration was observed in nonparenchymal cells or hepatocytes. Table A1. Alanine aminotransferase (ALT), creatinine, and aspartate aminotransferase (AST) in mice after 96 h administration of 3.7 MBq of 177 Lu DOTA-PD-L1-i (n = 3) or 3.7 kBq of 225 Ac-HEHA-PD-L1-i (n = 3) on day 5 after intravenous inoculation of HCC827 lung cancer cells (mean ± standard deviation). No significant differences (p < 0.05) in the ALT, creatinine, and AST values between mice treated with radiopharmaceuticals and those of the control group were found.